U.S. patent application number 10/240904 was filed with the patent office on 2003-03-27 for use of substances that act as cascade inhibitors of the raf/mek/erk signal cascade, for producing a medicament to treat dna and rna viruses.
Invention is credited to Ludwig, Stephan, Pleschka, Stephan.
Application Number | 20030060469 10/240904 |
Document ID | / |
Family ID | 7638027 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030060469 |
Kind Code |
A1 |
Ludwig, Stephan ; et
al. |
March 27, 2003 |
Use of substances that act as cascade inhibitors of the raf/mek/erk
signal cascade, for producing a medicament to treat dna and rna
viruses
Abstract
The invention consists in that substances acting as cascade
inhibitors of the Raf/MEK/ERK signaling path-way, in particular MEK
inhibitors, are used for the production of a drug for the
preventive and antiviral therapy against DNA and RNA viruses, in
particular against intranuclear-replicating negative strand RNA
viruses, for instance influenza or Borna disease viruses.
Inventors: |
Ludwig, Stephan; (Wurzburg,
DE) ; Pleschka, Stephan; (Giessen, DE) |
Correspondence
Address: |
Mark D Wieczorek
PO Box 70072
San Diego
CA
91267
US
|
Family ID: |
7638027 |
Appl. No.: |
10/240904 |
Filed: |
October 4, 2002 |
PCT Filed: |
April 5, 2001 |
PCT NO: |
PCT/DE01/01292 |
Current U.S.
Class: |
514/251 ;
514/456; 514/617; 514/646 |
Current CPC
Class: |
A61P 31/14 20180101;
A61K 31/352 20130101; A61K 31/166 20130101; A61P 31/20 20180101;
A61P 31/12 20180101; A61P 31/16 20180101; A61K 31/145 20130101 |
Class at
Publication: |
514/251 ;
514/456; 514/646; 514/617 |
International
Class: |
A61K 031/525; A61K
031/353; A61K 031/165; A61K 031/135 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 7, 2000 |
DE |
100 17 480.9 |
Claims
1. Substances characterized by that they act as cascade inhibitors
according to the invention, in particular MEK inhibitors, in
particular originating from the chemical substance classes of the
butadiene derivatives or flavin derivatives or benzamide
derivatives.
2. An application of cascade inhibitors according to the invention,
in particular of the chemical substance classes of the butadiene
derivatives or flavin derivatives or benzamide derivatives, in
particular MEK inhibitors, in particular: a)
2-(2-Amino-3-methoxyphenyl)-4-oxo-4H-(1)ben- zopyran,
1,4-Diamino-2,3-dicyano-1, b) 4-bis[2-aminophenylthio]butadiene, c)
2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benz-
amide, d) 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalene-4-on e) all
pre-stage substances, salts, prodrugs, derivatives or mixtures of
the afore-mentioned substances acting as cascade inhibitors, in
particular MEK inhibitors, for the production of a drug for the
prevention and treatment of infections by DNA and RNA viruses in
man and animal.
3. An application according to claim 2 for the treatment of
infections by negative strand RNA viruses.
4. An application according to claim 2 for the treatment of
infections by intranuclear-replicating negative strand RNA
viruses.
5. An application according to claim 2 for the treatment of
infections by influenza and Borna viruses.
Description
SPECIFICATION
[0001] The present invention is based on the first observation that
an infection with the intranuclear-replicating negative strand
viruses, in particular influenza A virus and Borna disease virus
(BDV), will lead to an activation of the Raf/MEK/ERK cascade, and
that surprisingly the inhibition of this cascade in particular by a
MEK inhibitor considerably inhibits the replication of this virus
group, without having a toxic effect on the cells.
[0002] An improved therapy against DNA and RNA viruses the
multiplication of which is dependent on the activity of the
Raf/MEK/ERK cascade, is preferably directed therefore to this
signaling pathway. It has been found that this signaling pathway is
blocked by the application of a non-toxic pharmacological
inhibitor, This non-toxic pharmacological inhibitor of the
Raf/MEK/ERK signaling pathway is according to the invention a
cascade inhibitor, in particular a MEK inhibitor.
PRIOR ART
[0003] Virus infections are a considerable risk for the health of
man and animal. In particular infections with the influenza A virus
still belong to the big epidemics of mankind and are responsible
year for year not only for a multitude of fatalities, but are also
an immense cost factor for the whole economy, for instance by
absence from work due to diseases [12].
[0004] Of equally important economic significance are infections
with the Borna disease virus (BDV), in particular infecting horses
and sheep, however also having been isolated from man already and
being connected with neurological diseases [3, 13].
[0005] The problem of controlling RN viruses is the adaptability of
the viruses caused by a high error rate of the viral polymerases,
thus the production of suitable vaccines and also the development
of antiviral substances being very difficult.
[0006] It has been shown that the application of antiviral
substances immediately directed against functions of the virus,
will very quickly lead to the selection of resistant variants,
after a mutation. An example for this is the anti-influenza agent
amantadine and the derivatives thereof being directed against a
transmembrane protein and leading within a few passages already to
the generation of resistant variants. The new anti-influenza
therapeutic agents inhibiting the influenza-viral surface protein
neuraminidase and being sold under the tradename RELANZA by Glaxo
Wellcome in Germany, also have also produced variants already in
patients [10]. Hopes being connected with this therapeutic agent
could therefore not be fulfilled.
[0007] Due to the in most cases small genomes and thus limited
coding capacity for functions being necessary for replication, all
viruses are to a large extent dependent on the functions of their
host cells. By influencing such cellular functions being necessary
for the viral replication, it is possible to affect in a negative
way the virus replication in the infected cell. There is no
possibility for the virus to replace the missing cellular function
by adaptation. An escape from the selection pressure by mutation is
here not possible. This could already be shown for the example of
the influenza A virus with relatively unspecific inhibiting
substances against cellular kinases and methyl transferases
[18].
[0008] It is the drawback in particular of these inhibiting
substances that they have a relatively unspecific and broad effect,
and that their cellular attacking points are only poorly defined.
They are therefore not suitable for use as therapeutic agents. This
is the problem; Until today, there are no inhibiting substances of
cellular enzymes having a selective effect at this point without
being toxic for the cell, as well as inhibiting the viral
replication in particular of RNA viruses, such as Borna viruses or
influenza A viruses.
[0009] With regard to the cellular processes induced after a virus
infection, it is found that a multitude or DNA and RNA viruses
activate in the infected host cell a defined signal transduction
pathway, the so-called Raf/MEK/ERK kinase cascade [2, 4, 14,
17].
[0010] This kinase cascade belongs to the most important signaling
pathways in the cell and plays an essential role in proliferation
and differentiation processes.
[0011] Growth-factor induced signals are transferred by successive
phosphorylation from the serine/theorine kinase Raf to the dual
specific kinase MEK (MAP kinase kinase/ERK kinase) and finally to
the kinase ERK (extracellular signal regulated kinase). Whilst as a
kinase substrate of Raf, only MEK is known, and the ERK isoforms
have been identified for MEK as the only substrate, ERK can
phosphorylate quite a number of substrates. Hereto belong for
instance the phosphorylation of transcription factors, which leads
to a direct modification of the cellular gene expression [5, 15,
20].
[0012] The investigation of this signaling pathway in cellular
decision processes has led to the identification of several
pharmalogical inhibitors, which inhibit the signaling pathway,
among other positions, on the level of MEK, i.e. at the
`bottleneck` of the cascade [1, 5, 7, 9].
[0013] The MEK inhibitor PD98059
(2-2'-amino-3'-methoxyphenyl)-oxanaphthal- ene-4-on [7] inhibits
the activation of MEK by the kinase Raf.
[0014] The MEK inhibitor U0126
(1,4-diamino-2,3-dicyano-1,4-bis[2-aminophe- nylthio]butadiene) has
been described as a substance partially inhibiting the activation
of AP-1 dependent gene expression [9] and the proliferation of T
cells [6].
[0015] In contrast to PD98059, the U0126 inhibits not only the MEK
activation, but also the activity of the kinase itself [8].
[0016] Finally, the MEK inhibitor PD184352 has been described
(2-(2-chloro-4-iodo-phenylamino)-N-cyclopropyl-methoxy-3,4-difluoro-benza-
mide) [19], which with oral administration in the mouse model could
efficiently inhibit the growth of colon carcinoma, without showing
any significant signs of toxicity up to a cumulating dose of 6 g/kg
body weight.
OBJECT OF THE INVENTION
[0017] The invention is based on the object to provide substances
for application in the prevention or therapy against
intranuclear-replicating negative strand viruses, such substances
not being immediately directed against functions of the virus, but
selectively inhibiting a cellular enzyme, and inhibiting via this
selective effect the viral replication of viruses.
[0018] Surprisingly, it has been found that this object can be
achieved by a cascade inhibitor according to the invention or in
particular by drugs containing a MEK inhibitor according to claim
1.
[0019] The cascade inhibitor according to the invention, in
particular a MEK inhibitor, is a substance characterized by that it
inhibits in a "cascade assay for inhibitors of the Raf/MEK/ERK
kinase signaling pathway" the signaling cascade in vitro and in an
"in viva MEK and MAP kinase assay" the signaling cascade in
vivo.
[0020] Cascade Assay for Inhibitors of the Raf/MEK/ERK Kinase
Signaling Pathway
[0021] For this cascade assay, the effect of inhibitors on the
Raf/MEK/ERK signaling pathway is measured by kinase-arranged
integration of radioactive 32P in the myelin basic protein (MBP) in
presence of a 6.times.histidine fusion protein of ERK (his-ERK) and
a glutathione S-transferase fusion protein of MEK (GST-MEK).
[0022] The reaction mixture contains the recombinant proteins in a
buffer of 20 mM HEPES, pH 7.4, 10 mM Mgcl2, 1 mM MnCl2, 1 mM EGTA
and 50 mM 32P-gamma-ATP in a total volume of 100 .mu.l. The
reaction takes 15 min at 30.degree. C. and is stopped by addition
of 20 .mu.l Laemmli buffer. The radioactive-marked proteins were
separated by SDS-PAGE and made visible by a phospho imager. Cascade
inhibitors were tested in a concentration of 5-20 .mu.l for their
inhibiting ability in this assay. In order to differentiate whether
a composition in this assay is a MEK or ERK inhibitor, the
substances are tested in a second experimental approach with MBP
and his-ERK under the above reaction conditions in presence of
GST-MEK. A composition being effective in the first approach and
having no effect in the second approach, is a MEK inhibitor. A
composition being effective in the second approach and having no
effect in the first approach, is an ERK inhibitor. A substance not
being effective in any of the two approaches, having an effect
however in the following in vivo MEK and MAP kinase assay, is a Raf
inhibitor. All described inhibitors are according to the invention
cascade inhibitors.
[0023] In Vivo MEK and MAP Kinase Assay
[0024] Cells were sown in 10 cm cell culture dishes and grow to 80%
confluence in cell culture medium with 10% fetal calf serum. The
serum was removed for 8-12 h from the cells. Then the addition of
the cascade inhibitors, in particular of the MEK inhibitors, is
made, 30 min before the mitogenic stimulation of the cells, for
instance with 100 ng/ml TPA or 100 ng/ml PDGF. After 10 min
incubation with the mitogenic stimuli, the cells are washed with
PBS and lysated in triton lysis buffer. (20 mM Tris pH 7.4, 50 mM
Na-.beta.-glycerol phosphate, 20 mM Na pyrophosphate, 137 mM NaCl,
10% (v/v) glycerin, 1% (v/v) triton X100, 2 mM EDTA, 1 mM Pefabloc,
1 mM Na-vanadate, 5 mM benzamidine, 5 .mu.g/ml Aprotinin, 5
.mu.g/ml Leupeptin). From these cell lysates, endogenous MEK is
immuno-precipitated with a MEK-specific antiserum and incubated in
an immune-complex kinase assay in presence of 32P-gamma-ATP, 0.1 mM
ATP and recombinant kinase-inactive his-ERK K>M as the substrate
protein at 30.degree. C. for 15 min in a buffer of 10 mM MGCl2, 25
mM .beta.-glycerol phosphate, 25 mM HEPES pH 7.5, 5 mM benzamidine,
0.5 ml DTT and 1 mM Na vanadate. Simultaneously, from the same
lysate is immuno-precipitated endogenous ERK with a specific ERK
antiserum and purified MBP under the same conditions as MEK. The
proteins are dissociated on a SDS-PAGE gel and visualized by means
of a phospho imager. A cascade inhibitor, in particular a MEK
inhibitor acts in this assay in an inhibiting way on the MEK
activation, as measured by the phosphorylation of his-ERK K>M,
as well as on the ERK activation, as measured by the
phosphorylation of MBP.
[0025] The application according to the invention of the cascade
inhibitors, in particular of the MEK inhibitors, relates in
particular to the following substances:
[0026] a) 2-(2-Amino-3-methoxyphenyl)-4-oxo-4H-(1)benzo-pyran (as
also described in WO 98/37881)
[0027] b)
1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenyl-thio]butadiene (short
designation: U0126)
[0028] c)
2-(2-chloro-4-iodo-phenylamino)-N-cyclopropyl-methoxy-3,4-difluo-
ro-benzamide) (short-form designation: PD18453)
[0029] d) 2-(2'-amino-3'-methoxyphenyl-oxanaphthalene-4-on
(short-form designation: PD98059)
[0030] e) substances characterized by that they act as cascade
inhibitors according to the invention and originate in particular
from the chemical substance classes of the butadiene derivatives or
flavin derivatives or benzamide derivatives,
[0031] f) all derivatives of the aforementioned substances acting
as cascade inhibitors, in particular MEK inhibitors,
[0032] g) further substances acting as cascade inhibitors, in
particular MEK inhibitors (pre-stage substances, salts or
"prodrugs" in the meaning of [11, 16] of the afore-mentioned
compositions or their derivatives, the effectiveness of which in
the cascade assay for inhibitors of the Raf/MEK/ERK signaling
pathway or in the "in vivo MEK and MAP kinase assay" is
proven).
[0033] The invention relates to the application of these substances
as drugs for patients being infected with a DNA or RNA virus, in
particular an intranuclear-replicating negative strand RNA virus,
for instance an influenza A virus or a Borna disease virus.
[0034] In another type of the application according to the
invention, it is suggested to use drugs comprising these substances
for the prevention of an infection with a DNA or RNA virus, in
particular an intranuclear-replicating negative strand RNA virus,
for instance an influenza A virus or a Borna disease virus.
[0035] The term patient relates equally to human beings and
vertebrates. Thus the drugs can be used in human and veterinary
applications. The therapeutically effective substances of the
present invention are administered to the patients as part of a
pharmaceutically acceptable composition either in an oral, rectal,
parenteral-intravenous, intramuscular or subcutaneous,
intracisternal, intravaginal, intraperitoneal, intravascular, local
(powder, ointment or drops) or spray form.
[0036] Pharmaceutically acceptable compositions may contain the
modifications as salts, esters, amides and "prodrugs", as far as
they will not, after a reliable medical evaluation, cause excessive
toxicity, irritations or allergic reactions of the patient.
[0037] The term "prodrug" relates to compositions being transformed
for a better reception, as for instance by hydrolysis in blood. A
detailed discussion is given in [11] and [16].
[0038] Dosing types for the local administration of the composition
of the invention include ointments, powder, sprays or inhalation
means. The active component is mixed under sterile conditions with
a physiologically acceptable carrier and possible preservatives,
buffers or driving means, depending on the necessity.
EXAMPLES
[0039] The example 1 shows for the MEK inhibitor U0126 that with
increasing concentration of the inhibitor U0126 in the cell culture
medium, the number of the newly generated infectious influenza A
virus particles is significantly reduced.
[0040] For the multiplication of influenza A viruses, permissive
eukaryotic cell cultures (Madine-Darby canine kidney (MDCK) cells),
were washed in parallel approaches having equal cell counts with a
physiological salt solution and infected with an equal amount of
the infectious influenza A virus stem WSN-HK(reassortant having
seven gene segments of influenza stem A/WSN/33 and the NA gene of
influenza stem A/HK/8/68), in a ratio of 0.0025 infectious virus
particles per cell for one hour at room temperature.
[0041] 30 min before the infection, the MDCK cells are incubated in
a suitable cell culture medium being reacted in different
concentrations with the MEK inhibitor U0126 (0 .mu.M, 30 .mu.M, 40
.mu.M, 50 .mu.M dissolved in DSMO) at 37.degree. C. and 5% CO2. As
a solvent reference, MDCK cells were incubated with cell culture
medium reacted with the corresponding various amounts of DMSO.
During the infection, the MEK inhibitor U0126 or DMSO as a solvent
is added to the inoculum in the corresponding concentrations.
[0042] Subsequently, the inoculum is removed, and the infected
cells are incubated in a suitable cell culture medium being reacted
in different concentrations with the MEK inhibitor U0126 (0 .mu.M,
30 .mu.M, 40 .mu.M, 50 .mu.M dissolved in DSMO) for 48 h at
37.degree. C. and 5% CO2. As a solvent reference, MDCK cells were
incubated with cell culture medium reacted with the corresponding
various amounts of DMSO. 24 hours after the infection, 200 .mu.l of
the medium supernatant were removed, and the same volume of
inhibitor or DMSO-containing cell culture medium were re-added to
the medium supernatant. After 48 h, another sample was taken. The
cell culture supernatants of the respective samples for the 24 and
the 48 h value are examined to conventional virological methods for
the amount of hemagglutinating units (HA titer) representing the
total production of virus particles, and for the amount of newly
generated infectious virus particles (plaque assay on MDCK
cells).
[0043] As a result, it can be found in such an experimental
approach that with increasing concentration of the MEK inhibitor
U0126 the number of newly generated infectious virus particles is
significantly reduced (approx. 80% for 50 .mu.M U0126) in the cell
culture medium, compared to the reference approach without MEK
inhibitor U0126 or the solvent references, respectively. The
macroscopic examination of MDCK cells treated with corresponding
concentrations of DMSO or MEK inhibitor U0126 dissolved in DMSO, as
well as a cytotoxicity examination by means of propidium iodide
staining show that neither solvent nor inhibitor have a significant
cytotoxic effect on the cells.
[0044] The example 2 shows that with increasing concentration of
the MEK inhibitor U0126 in cell culture medium also the number of
newly generated infectious Borna disease viruses particles is
significantly reduced.
[0045] Cells pre-treated with inhibitor are infected with BDV, and
the spreading of the infection is observed in an indirect
immunoflorescence against the viral nucleoprotein. After a one-time
administration of 25 .mu.M MEK inhibitor (U0126), no virus foci are
visible after a cultivation time of 7 days, but only individual
infected cells. After an administration of 12.5 .mu.M kinase
inhibitor (U0126), the effect is not clear anymore, and after an
administration of 6 .mu.M kinase inhibitor (U0126), no difference
of the virus foci can be found compared to untreated infectious
reference cells. The inhibitor acts therefore in a dosage-dependent
manner on the level of the virus replication.
[0046] The inhibitory effect of the MEK inhibitor (U0126) in the
described applications shows that the cascade inhibitors, in
particular MEK inhibitors, can be used as antiviral agents against
influenza and Borna viruses in particular, however also against RNA
and DNA viruses, for which a dependence of the viral multiplication
on the activity of the Rat/MEK/ERK cascade exists. The signaling
path is herein according to the invention the aim of the antiviral
therapy and is preferred by application of a non-toxic
pharmacological cascade inhibitor, in particular a MEK
inhibitor.
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